T-cell receptor ligands and methods of using same

ABSTRACT

The present invention concerns TCR ligands with immunomodulatory properties, as well as methods of identifying such ligands and of using such ligands to modulate T cell effector responses.

This application is a divisional of U.S. patent application Ser. No.08/004,936, filed Jan. 15, 1993 and now U.S. Pat. No. 5,837,477.

TECHNICAL FIELD OF THE INVENTION

The present invention concerns T cell receptor ligands, moreparticularly novel peptide-major histocompatibility complex class IImolecule ligands, with novel immunomodulatory properties, as well asmethods of using such ligands to modulate T cell effector responses, andmethods to identify such ligands.

BACKGROUND OF THE INVENTION

Both humoral and cellular immune responses are essential components ofdefenses against pathogenic bacteria, viruses, and parasites. Keyplayers in the immune response, called T lymphocytes, control cellularimmunity by virtue of their ability to discriminate between a particularantigen and its close relative. This remarkable antigen specificity ofthe T lymphocyte responses is due to the presence on the T cell surfaceof clonally distributed, immunoglobulin-like T cell receptors (TCR)consisting of two non-identical glycosylated polypeptide chains, calledα and β. T lymphocytes also express on their surface glycoproteins thatare markers of different stages and types of T cell maturation (e.g.,T3, T4, and T8 glycoproteins present on CD3⁺, CD4⁺ and CD8⁺ T cells,respectively), which may mediate or augment specific T cell functions.

TCRs interact with antigens that have been processed by the antigenpresenting cell (APC) to unfold or cleave the protein into peptidefragments, and are presented on the cell surface as part of a complexwith a molecule encoded by genes within the major histocompatibilitycomplex (MHC). CD8⁺ and CD4⁺ T lymphocytes interact with peptides boundto the polymorphic region of MHC class I or class II molecules,respectively (Townsend et al., Ann. Rev. Immunol., 7, 601-24 (1989);Rothbard et al., Ann. Rev. Immunol., 9, 527-65 (1991)). The TCR alsointeracts with proteins that have the capacity to generate intracellularsecond messenger signals that are essential to triggering T cellactivation (Ashwell et al., Ann. Rev. Immunol., 8, 139-67 (1990)), orinduction of T cell proliferation or differentiation. For example,recent data show that the CD3 γ, δ, ε: ζ/^(n) complex stably associatedwith the TCR consists of at least two separate signal transductionmodules that initiate second messenger cascades (Letourneur et al.,Proc. Natl. Acad. Sci. USA, 88, 8905-09 (1991); Wegener et al., Cell,68, 83-95 (1992)) which include primary events such as tyrosinephosphorylation (Samelson et al., Cell, 46, 1083-90 (1986)) andsecondary events such as PIP₂ hydrolysis and elevation of Ca⁺⁺ !_(i)(Weiss et al., Proc. Natl. Acad. Sci. USA, 81, 4169-73 (1984); June etal., J. Immunol., 144, 1591-99 (1990)).

The effect of these TCR-mediated biochemical events on the T cell isinfluenced by independent receptor-ligand interactions that may generatedifferent types of signals from those evoked by the TCR-ligandinteraction (Weaver et al., Proc. Natl. Acad. Sci. USA, 85, 8181-85(1988); Mueller et al., J. Immunol., 144, 3701-09 (1990); Linsley etal., J. Exp. Med., 173, 721-30 (1991); Koulova et al., J. Exp. Med.,173, 759-62 (1991); Vandenberghe et al., J. Exp. Med., 175, 951-60(1992)). Studies manipulating the potential of the APC to provideco-stimulation (Quill et al., J. Immunol., 138, 3704-12 (1987); Otten etal., Science, 251, 1228-31 (1991)) have separated effector activities ofCD4⁺ and CD8⁺ T cells into activities that do (e.g., cytokineinterleukin (IL)-2 production) and do not (e.g., cytokine IL-3production and cell killing) require co-stimulatory signals.

The participation of co-stimulatory signals in control of production ofIL-2 (Jenkins et al., Immunol. Rev., 95, 113-35 (1987)), the cytokinethat is primarily responsible for clonal expansion following T cellactivation, is indicated by the finding that metabolically-inactivatedcells bearing ligands for the TCR present on CD4⁺ cells are unable toeffectively stimulate IL-2-dependent T cell proliferation (Bach et al.,Immunol. Rev., 35, 76-96 (1977); Germain, J. Immunol., 127, 1964-66(1981); Jenkins et al., J. Exp. Med., 165, 302-19 (1987)), whichsuggests there is a critical `second signal` missing in theseinactivated cells that operates independently of TCR-regulated secondmessenger generation or augmentation of TCR occupancy (Mueller et al.,J. Immunol., 142, 2617-28 (1989a)). Several candidate receptor-ligandpairs have been suggested for the co-stimulatory pathway, such as thebinding of B7 surface protein on the APC by CD28 on the responding Tcell (Freeman et al., J. Exp. Med., 174, 625-31 (1991); Gimmi et al.,Proc. Natl. Acad. Sci. USA, 88, 6575-79 (1991); Koulova et al., J. Exp.Med., 173, 759-62 (1991); Linsley et al., J. Exp. Med., 173, 721-30(1991); Reiser et al., Proc. Natl. Acad. Sci. USA, 89, 271-75 (1992);Vandenberghe et al., J. Exp. Med., 175, 951-60 (1992)), and therecognition of the heat-stable antigen on the APC by an uncharacterizedT cell counter-receptor (Kay et al., J. Immunol., 145, 1952-59 (1990);Liu et al., J. Exp. Med., 175, 437-45 (1992)).

Activation of the T cell is initiated when some adequate number of TCRsare aggregated at the interface between the T cell and the APC (Singer,Science, 255, 1671-77 (1992); Matis et al., Proc. Natl. Acad. Sci. USA,80, 6019-23 (1983a); Ashwell et al., J. Immunol., 136, 757-68 (1986)).The extent of receptor-ligand aggregation depends on the number ofavailable receptors on the T cell, the number of available ligands,i.e., peptide-MHC molecule complexes, on the APC, and the affinity ofthe TCR for the ligand. When a high level of peptide-MHC moleculecomplexes on the APC fails to induce T cell activation, it is believedthis is due to a low affinity of the TCR for the ligand, which preventsreceptor occupancy from exceeding the threshold needed for secondmessenger generation within the T cell (Fiering et al., Genes Dev., 4,1823-34 (1990).

This affinity-based occupancy model predicts that in the presence ofintact, metabolically-active APC capable of delivering co-stimulatorysignals, peptide-MHC molecule complexes will be of two types: (1)agonists that can induce full T cell activation, and (2) non-agoniststhat do not induce T cell activation because of low affinity of the TCRfor the peptide-MHC molecule complex, which prevents the number ofoccupied TCR from reaching the triggering threshold level (Matis et al.,Proc. Natl. Acad. Sci. USA, 80, 6019-23 (1983a)). Recently, this modelhas been challenged by findings showing that substitution of a singleresidue in the peptide antigen for the TCR on a mouse Th2 cloneprevented stimulation of proliferative responses, while permitting IL-4cytokine production (Evavold et al., Science, 252, 1308-10 (1991)). Thisindicates that contrary to predictions of the affinity-based occupancymodel, certain ligands can stimulate T cell second messenger generationwithout evoking the full repertoire of effector responses. Moreover,peptide analog-MHC molecule complexes have been described which inhibitthe IL-2 response of the T cell response by TCR antagonism, orcompetition with wild-type ligand for binding to the TCR (De Magistriset al., Cell, 68, 625-34 (1992)). It has been reported that theinhibitory complexes were pure TCR antagonists which lacked capacity togenerate intracellular signals (De Magistris et al., Cell, 68, 625-34(1992)). This finding of an absence of second messenger generationdespite fully occupied TCRs is also not predicted by the affinity basedoccupancy model.

The present invention is predicated on the unexpected discovery thatthere exist TCR ligands which exhibit selective antagonist properties(referred to herein as "selective antagonists") and which may alsoconcurrently exhibit agonist properties (referred to herein as "mixedagonists-antagonists"). Specifically, peptide-MHC molecule complexeshave been identified which interact with the TCR to actively andselectively inhibit IL-2 production by a mouse T cell clone, withoutpreventing IL-3 production, IL-2Rα upregulation, or cell sizeenlargement induced by a TCR agonist. Since these new TCR ligands areable to selectively modulate certain T cell effector activities in aTCR-specific manner, they can be considered selective antagonists. Theseselective antagonists differ from the partial agonists described inEvavold et al., Science, 252, 1308-10 (1991), in that the selectiveantagonists of the present invention actively inhibit certain effectorresponses as opposed to simply failing to stimulate these responses.These selective antagonists differ from the complete antagonistsdescribed in De Magistris et al., Cell, 68, 625-34 (1992), in thatunlike the complete antagonists, the selective antagonists of thepresent invention have been shown to selectively inhibit certaineffector responses, without affecting others, and act without preventingall T cell signaling.

These results suggest there may be two distinct classes of inhibitorypeptide-MHC molecule complexes: selective antagonists and completeantagonists. While members of the latter class would preventintracellular messenger generation in the T cell by removing TCRs fromthe functional pool and precluding any effector responses, members ofthe former class would interfere with certain effector activities basedon qualitative differences in requirements for intracellular signalling,possibly, but not necessarily, related to the co-stimulation dependenceof the analyzed functions.

The properties of the TCR ligands of the present invention haveimportant implications for models of thymic selection and peripheral Tcell activation and provide new pharmacological approaches to thetreatment of autoimmune disease, to the problems of graft rejection, andin vaccine design. Moreover, the present invention described hereinenables the identification, characterization, development, andutilization of the TCR ligands of the present invention.

Consequently, it is an object of the present invention to provide a TCRligand which inhibits at least one T cell effector response evoked byfully active peptide-MHC molecule complexes available to responding Tcells, without necessarily inhibiting all other effector responses ofthe T cells. It is a related object of the present invention to providea TCR ligand which inhibits at least one T cell effector response evokedby fully active peptide-MHC molecule complexes available to responding Tcells and which does not substantially inhibit at least one other T celleffector response. It is another object of the present invention toprovide a TCR ligand which inhibits co-stimulation dependent T celleffector responses evoked by fully active peptide-MHC molecule complexesavailable to responding T cells and which does not block co-stimulationindependent T cell effector responses under the same conditions. It isyet another object of the present invention to provide TCR ligands whichare selective antagonists and mixed agonists-antagonists. It is afurther object of the present invention to provide a method ofidentifying, as well as preparing, such TCR ligands and of providingimproved methods of modulating T cell effector response utilizing suchTCR ligands.

These and other objects and advantages of the present invention, as wellas additional inventive features, will be apparent from the descriptionof the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a TCR ligand which substantially inhibitsat least one T cell effector response evoked by fully active peptide-MHCmolecule complexes available to responding T cells without necessarilysubstantially inhibiting, and preferably not substantially inhibiting,at least one other T cell effector response evoked by fully activepeptide-MHC molecule complexes available to responding T cells. Thepresent invention further provides for a TCR ligand which inhibitsco-stimulation dependent T cell effector responses evoked by fullyactive peptide-MHC molecule complexes available to responding T cellsand which does not block co-stimulation independent T cell effectorresponses under the same conditions. The present invention provides TCRligands which are selective antagonists and mixed agonists-antagonists.

The present invention additionally provides methods of using such TCRligands to modulate T cell effector responses, as well as methods toidentify, and develop candidate members of, such TCR ligands.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the response of 3C6 Th1 cells to pigeon cytochrome C(PCC) peptide PCC 81-104 presented by EαEβ^(k) molecules with differentmutations in the βHV3 region as measured by stimulation of IL-2production. FIG. 1A demonstrates alloreactivity of 3C6 Th1 cells to themutated EαEβ^(k) molecules in the absence of peptide. FIG. 1Bdemonstrates the response of 3C6 Th1 cells to MHC EαEβ molecules withβ72^(s),75^(s),79^(s) (sss) or β75^(s),79^(s) (kss) mutant chains in thepresence of PCC 81-104 peptide.

FIG. 2 sets forth the inhibitory effect of different peptides onalloreactive stimulation of IL-2 production in 3C6 Th1 cells by MHCmolecules with kss Eβ chains and shows that the inhibitory effect isrelated to peptide ability to be recognized by the 3C6 receptor. FIG. 2Ademonstrates binding of the peptides PCC 81-104, PCC 81-104 99Q!, DASP,and HEL 46-61 to transfected L cells expressing MHC molecules with kssEβ chains as measured by competition for binding of biotinylated-DASP(25 μM). Data are expressed as percent inhibition at each competitorconcentration. FIG. 2B demonstrates the response of 3C6 Th1 cells to thepeptides PCC 81-104, PCC 81-104 99Q!, and DASP presented by an L celltransfectant expressing MHC molecules with wild-type Eβ^(k) kkk chainsas measured by percent of maximal stimulation of IL-2 production. FIG.2C demonstrates the response of 3C6 Th1 cells to the peptides PCC81-104, PCC 81-104 99Q!, and DASP presented by an L cell transfectantexpressing MHC molecules with kss Eβ chains as measured by percent ofstimulation of IL-2 production in the absence of added peptide.

FIG. 3 sets forth the ability of a peptide to bind MHC molecules withkss Eβ chains and shows that this binding ability does not correlatewith its ability to inhibit alloreactive stimulation of IL-2 productionin 3C6 Th1 cells. FIG. 3A demonstrates direct binding of thebiotinylated peptides PCC 88-104 and HEL 81-96 to an L cell transfectantexpressing MHC molecules with kss Eβ chains. Data are expressed as meannet fluorescence. FIG. 3B demonstrates the effect of the biotinylatedpeptides PCC 88-104 and HEL 81-96 on alloreactive stimulation of IL-2production by an L cell transfectant expressing MHC molecules with kssEβ chains. Data are expressed as percent of IL-2 production obtained inthe absence of added peptide.

FIG. 4 sets forth the inhibitory effect of PCC 81-104 and shows thathigh dose inhibition does not account for the inhibitory effect of PCC81-104 on alloreactive stimulation of IL-2 production in 3C6 Th1 cellsby L cells expressing MHC molecules with kss Eβ chains. FIG. 4Ademonstrates the response of 3C6 Th1 cells to PCC 81-104 presented by anL cell transfectant expressing MHC molecules with wild-type kkk Eβchains as measured by percent stimulation of IL-2 production andproliferation. FIG. 4B demonstrates the response of 3C6 Th1 cells to PCC81-104 presented by an L cell transfectant expressing MHC molecules withkss Eβ chains as measured by percent of IL-2 production andproliferation obtained in the absence of added peptide.

FIG. 5 sets forth the inhibitory effect of PCC 81-104 and shows that thePCC 81-104 peptide-mediated inhibition of alloreactive stimulation ofIL-2 production in 3C6 Th1 cells is selective and does not prevent otherTCR-dependent activation events. FIG. 5A demonstrates two-color flowcytometry profiles of 3C6 cells co-cultured for 24 hours withtransfected L cells expressing either MHC molecules with wild-type kkkor mutant kss Eβ chains. The left panel shows anti-IL-2 receptor α chain(7D4) staining of Thy-1 (G7)-negative and positive cells. The Thy-1protein is a marker for T lymphocytic 3C6 Th1 cells. The right panelshows size (SSC) of the G7-positive or 3C6 Th1 cells. The Eβ chain ofthe MHC molecule expressed by the L cell as well as the presence orabsence of PCC 81-104 are indicated in each panel. FIG. 5B demonstratesthe relative change in IL-2Rα expression (IL-2Rα) for cells included inthe gate 1 subset of Thy-1⁺ cells in FIG. 5A (i.e., 3C6 Th1 cells), insize (SSC), in IL-2 production (IL-2), and in IL-3 production (IL-3).While IL-2Rα was determined by comparing cultures with transfectantsexpressing MHC molecules with kss Eβ chains in the absence and presenceof PCC 81-104, SSC was calculated as change in percent of cells in gate1 of the right panel of FIG. 5A by comparing co-cultures withtransfectants expressing the kss Eβ chain in the absence and presence ofPCC 81-104, and IL-2 and IL-3 production were determined by comparingrelative production in co-cultures with transfectants expressing the kssEβ chain in the absence and presence of PCC 81-104. FIGS. 5C and 5Ddemonstrate reverse transcription-PCR analysis of RNA extracted fromco-cultures of 3C6 Th1 cells and transfected L cells expressing MHCmolecules with kss Eβ chains in the absence or presence of PCC 81-104 (1μM). While FIG. 5C shows ethidium staining of PCR products, FIG. 5Dshows shows the calculated relative amounts of IL-2 and IL-3 mRNA.

FIG. 6 sets forth the antigen dose-response patterns of stimulated IL-2and IL-3 production by 3C6 Th1 cells to peptide presented on live orfixed L cell transfectants expressing MHC molecules with wild-typeEβ^(k) kkk chains. Results are expressed as the percent of the maximumIL-2 or IL-3 response, respectively, obtained with live or fixed APC.

FIG. 7 sets forth the inhibitory effect of PCC 81-104 and shows that PCC81-104 does not inhibit the stimulation of IL-2 production and canincrease the stimulation of IL-3 production in C6E1 hybridoma cells by Lcell transfectants expressing MHC molecules with kss Eβ chains. FIG. 7Ademonstrates the response of the C6E1 hybridoma cells to PCC 81-104peptide presented by L cells expressing MHC molecules with wild-type kkkEβ chains and measured by stimulation of production of IL-2 and IL-3.Data are expressed as percent of maximal stimulation for IL-2 and forIL-3. FIG. 7B demonstrates the response of the C6E1 hybridoma cells toPCC 81-104 peptide presented by L cells expressing MHC molecules withkss Eβ chains and measured by stimulation of production of IL-2 andIL-3. Data are expressed as percent of the response obtained in theabsence of PCC 81-104 peptide.

FIG. 8 sets forth the inhibitory effect of PCC 81-104 and shows thatantibody cross-linking of CD28 on 3C6 Th1 cells does not preventselective PCC 81-104 peptide-mediated inhibition of alloantigenstimulated production of IL-2. FIG. 8A demonstrates stimulation of IL-2and IL-3 production in 3C6 Th1 cells by an L cell transfectantexpressing MHC molecules with kss Eβ chains in the presence ofmonoclonal antibody (mAb) directed against CD28 (1:250 dilution ofascites). Data are expressed as percent of the response obtained in theabsence of the anti-CD28 mAb. FIG. 8B demonstrates the effect ofantibody-mediated cross-linking of CD28 on stimulation of IL-2 and IL-3production in 3C6 Th1 cells by an L cell transfectant expressing MHCmolecules with kss Eβ chains in the absence and presence of 1 μM PCC81-104 peptide. Data are expressed as percent of the response obtainedin the absence of both the mAb and the PCC peptide.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns TCR ligands with immunomodulatoryproperties. Specifically, the present invention provides TCR ligandscapable of interfering with the elicitation of T cell effectorresponses. Ligands comprised of mutated MHC class II molecules verifiedthat a single alteration in the structure of the TCR ligand onphysiologically intact APC can inhibit at least one T cell effectorresponse evoked by fully active peptide-MHC molecule complexes availableto responding T cells. The present findings also reveal a remarkable andpreviously undisclosed class of peptide-MHC molecule complexes which,unlike the previously described complete antagonists, have been shown tosubstantially inhibit at least one T cell effector response (e.g.,stimulated production of IL-2) without substantially inhibiting other Tcell effector responses (e.g., induction of IL-3 secretion, IL2Rαup-regulation, and size-enlargement responses).

The present invention provides new classes of TCR ligands calledselective antagonists and mixed agonists-antagonists. The selectiveantagonists and mixed agonists-antagonists differ from the previouslydescribed partial agonists in that, rather than merely failing tostimulate certain effector responses, these new TCR ligands can activelyinhibit effector responses (e.g., stimulated IL-2 production) that wouldotherwise be evoked by agonist ligands on the same APC.

The selective antagonist of the present invention is a TCR ligand whichsubstantially inhibits at least one T cell effector response evoked byfully active peptide-MHC molecule complexes available to responding Tcells and preferably, but not necessarily, does not substantiallyinhibit at least one other T cell effector response evoked by fullyactive peptide-MHC molecule complexes available to responding T cells(i.e., that particular T cell or a T cell population).

The mixed agonist-antagonist of the present invention is a TCR ligandwhich substantially inhibits at least one T cell effector responseevoked by fully active peptide-MHC molecule complexes when madeavailable to responding T cells and which, while inhibiting one or moreT cell effector responses, stimulates one or more different T celleffector responses. The mixed agonist-antagonist preferably, but notnecessarily, does not substantially inhibit at least one other T celleffector response evoked by fully active peptide-MHC molecule complexeswhen made available to responding T cells.

Both the selective antagonist and the mixed agonist-antagonist may also,and preferably do, inhibit co-stimulation dependent T cell effectorresponses evoked by fully active peptide-MHC molecule complexesavailable to responding T cells and without blocking co-stimulationindependent T cell effector responses under the same conditions. Severalobservations described herein, such as the selective inhibitory effectof peptide ligand on IL-2 production, the dramatic effect of fixation ofAPC on the IL-2 but not IL-3 antigen dose-response relationship, and thefailure of peptide ligand to inhibit IL-2 production by a T cellhybridoma, confirm that the peptide-mutant MHC molecule complexes caninhibit co-stimulation dependent T cell effector responses evoked byfully active peptide-MHC molecule complexes by interfering with theproduction of, or response to, co-stimulatory signals.

Moreover, soluble antibody directed against CD28 affected the 3C6response to alloantigen in a manner similar to addition of PCC peptide,in that IL-3 responses were maintained in the face of inhibition of IL-2production. Antibody-mediated cross-linking of CD28 on 3C6 Th1 cellsabrogated the inhibition of alloresponses by soluble CD28 and slightlyincreased the IL-3 response. This verifies effective stimulation of theCD28 pathway. However, cross-linking of the CD28 molecules did notprevent the inhibition of IL-2 production mediated by PCC-mutant MHCclass II molecule complexes. These observations suggest that aninteraction between B7 and CD28 may be necessary but not sufficient forobservation of the IL-2 alloresponse. Alternatively, it is also possiblethat the intracellular signals delivered through CD28 are not effectivewhen the 3C6 TCR is engaged with the PCC peptide-mutant EαEβ complexes,which would suggest generation of dominant-negative intracellularmessengers by the incomplete agonists. Irrespective of whether the siteof defective signalling is the T cell or the APC, the ability ofrelatively small numbers of ligands in accordance with the presentinvention to interfere with T cell effector responses in the presence ofcomplete agonist ligand implies that introduction of ineffectiveTCR-ligand complexes into the signalling assemblies at the T cell-APCinterface interferes in a nonlinear way with signal generation.

In addition to providing novel TCR ligands, the present inventionprovides for a method of identifying such TCR ligands. Theidentification method for the selective antagonists comprises contactingT cells with an agonist capable of effecting known T cell effectorresponses and a candidate TCR ligand and determining whether thecandidate TCR ligand substantially inhibits at least one T cell effectorresponse. The present inventive method preferably further comprisesdetermining whether the candidate TCR ligand substantially inhibits atleast one T cell effector response while not substantially inhibiting atleast one other T cell effector response. In carrying out theidentification method, the T cells may be contacted with the agonist andcandidate TCR ligand in any suitable manner. Preferably, the T cells aresimultaneously contacted with the agonist and the candidate TCR ligandby contacting the T cells with a mixture of the agonist and thecandidate TCR ligand. While the mixture of the agonist and the candidateTCR ligand may be formed in any suitable manner, the mixture of agonistand candidate TCR ligand is preferably formed by contacting MHCmolecules with a first peptide to form the agonist and then with asecond peptide to form the candidate TCR ligand. Alternatively, the Tcells are contacted with the agonist and then the T cells and theagonist are contacted with the candidate TCR ligand.

The identification method for the mixed agonists-antagonists is the sameas that for selective antagonists with the additional step of separatelycontacting T cells with a candidate TCR ligand, with and without anagonist capable of effecting known T cell effector responses, andevaluating T cell effector responses to the candidate TCR ligand aloneas well as comparing the inhibitory effect of the candidate TCR ligandon at least one of the known T cell effector responses to agonists.

The present invention not only comprises methods of identifying the TCRligands of the present invention, but also contemplates methods ofpreparing candidate TCR ligands of the present invention. The method ofpreparing candidate TCR ligands as possible selective antagonists ormixed agonists-antagonists of the present invention comprisesidentifying a peptide which binds to MHC molecules to form a complexwhich can evoke a T cell effector response, determining which residuesof the peptide can be substituted so as not to affect binding to the MHCmolecules, determining which of the non-binding-effect residues of thepeptide affect recognition of the peptide-MHC molecule complexes by Tcells, substituting the non-binding-effect/recognition-effect residuesof the peptide to form substituted peptides, and screening thesubstituted peptides to identify those substituted peptides which haveless agonistic effect or a distinct spectrum of agonist effects (e.g.,with respect to different agonist effects) as compared to theunsubstituted peptides as candidate TCR ligands. The substitutedpeptide-MHC molecule ligands can then be further screened by contactingT cells with an agonist capable of effecting known T cell effectorresponses and one of the candidate TCR ligands and determining whetherthe candidate TCR ligand substantially inhibits at least one T celleffector response or, preferably, whether the candidate TCR ligandsubstantially inhibits at least one T cell effector response while notsubstantially inhibiting at least one other T cell effector response.

While the present inventive methods of preparing candidate TCR ligands,and identifying those TCR ligands of the present invention, haveapplicability in developing TCR ligands useful in managing autoimmunediseases and problems of graft rejection, these present inventivemethods also have applicability in vaccine design, especially in casesin which some components of an immune response have pathological ratherthan beneficial effects. In particular, selective antagonists can bedesigned with the capacity to selectively guide the immune responsealong certain pathways and avoid vaccine-induced immune responses thatcause pathology. For example, the split in cytokine production seen withsuch selective antagonists can also be used to deviate immune responsesfollowing vaccination away from those that sometime potentiate diseaseupon subsequent infection and towards those that are highly protective.

The present invention also comprises a method of using the T cellligands described herein to modulate T cell effector responses bycontacting T cells with the TCR ligands of the present invention. Moreparticularly, the present invention includes a method of modulating theimmune response of a host by administering to the host the TCR ligandsof the present invention.

The ability of the presently described TCR ligands to dominantlyinterfere with T cell effector function has implications for thecreation of new approaches to autoimmune disease treatment, problems ofgraft rejection, and vaccine design.

The presently described ligands able to engage TCRs and deviate theresponse of T lymphocytes to simultaneously available agonist ligandsfor the same TCR can be employed as highly selective agents to interruptthe effector activities of autoimmune T cells responding toself-antigen-MHC molecule complexes or alloimmune T cells responding totissue graft antigens. Previous approaches to the interruption ofautoimmune disease processes or graft rejection caused by T cellactivity have involved use of general immunosuppressive agents such assteroids or cyclosporin A/FK506, cytotoxic agents such ascyclophosphamide or methotrexate, and peptides that can physically blockthe MHC molecule binding site and prevent presentation of the peptidesinvolved in the disease or rejection process. These methods are fraughtwith numerous problems. For example, general immunosuppressives ornonselective cytotoxic agents leave the patient more prone to infectionand to development of malignancies, and are associated with a high levelof undesirable side-effects, such as renal and liver damage. Similarly,blocking the MHC binding site requires massive amounts of material toachieve the necessary quantitative blocking effect, may be accompaniedby induction of undesirable strong immune responses to the blockingagent itself, and calls for continuous treatment since the effect on theT cells is not prolonged. Moreover, the evidence does not support thatpeptides which block the MHC binding site can affect presentation ofself-antigens that are pre-associated with MHC molecules, which would benecessary for treatment when active disease is already present, or withallogeneic grafts.

The complete antagonists described in De Magistris et al., Cell, 68,625-34 (1992), avoid several of these problems as the antagonists areimmunologically specific and thus affect only a small subset of T cellsrelevant to disease. This reduces chances of adverse systemic ororgan-specific side-effects, and lessens the amount of materialnecessary for administration as compared to the MHC molecule blockingstrategy. However, these complete antagonists employed have been claimednot to generate intracellular signals (De Magistris et al., Cell, 68,625-34 (1992)), which means that any effects of administration would betransient. In distinct contrast, the present inventive approach isspecific and also may allow longer-lasting inactivation of autoimmune Tcells, as the novel TCR ligands described herein appear to requireintracellular second messenger generation and selectively inhibit only asubset of the responses of the T cell. Additionally, administration ofthe novel TCR ligands of the present invention could potentially driveautoimmune T cells into a long-term unresponsive state. This is becauseT cell signalling in the absence of co-stimulation frequently leads to astate of unresponsiveness termed anergy (Schwartz, Science, 248, 1349-56(1990)). Therefore, a selective antagonist or mixed agonist-antagonistin accordance with the present invention may potentially be able toblock ongoing autoimmune T effector activity, as might a complete MHCblocking peptide, and administration of such a TCR ligand may lead to alasting decrease in autoimmune disease due to anergy induction among theself-reactive T cells. In this fashion, ligand administration coulddiminish or slow the progression of the autoimmune disease process, orlengthen the survival of grafts. This would be accomplished with few orno side-effects, due to the extreme specificity of the drug for only thedisease-causing or graft-rejecting T cells.

Both in vitro and in vivo applications are contemplated in the contextof the present invention. It will be recognized that for the differentapplications, the TCR ligand may be employed in any suitable form andmay be formed in situ. Thus, the present invention contemplates theformation of a suitable TCR ligand through use of a suitable peptide,MHC molecule, or peptide-MHC molecule complex, which may be used aloneor in appropriate association with other agents, and which may beintroduced by addition to cells, expression in cells, presentation onthe surface of APC, or introduction by any other appropriate means orcombination of means. The TCR ligand or ligand component may be presentin a pharmaceutical composition in any suitable quantity. Thepharmaceutically acceptable excipients described herein, for example,vehicles, adjuvants, carriers, or diluents, are readily available to thepublic.

As regards these applications, the present inventive method includes theadministration to an animal, particularly a human, of a therapeuticallyeffective amount of one or more of the aforementioned TCR ligands orligand components as an active agent effective in the treatment of anycondition involving the desirability of modulating T cell effectorresponses, particularly autoimmune disease and problems of graft-hostrejection. One skilled in the art will appreciate that suitable methodsof administering a compound of the present invention to an animal areavailable, and, although more than one route can be used to administer aparticular compound, a particular route can provide a more immediate andmore effective reaction than another route. Pharmaceutically acceptableexcipients are also well-known to those who are skilled in the art, andare readily available. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition of thepresent invention. The following methods and excipients are merelyexemplary and are in no way limiting. However, pharmaceuticallyacceptable excipients which do not interfere with the desired effect onthe T cell effector response are, of course, preferred.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

The compounds of the present invention, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They may alsobe formulated as pharmaceuticals for non-pressured preparations such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, the TCR ligands or ligand components employed in thepresent invention may be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a prophylacticor therapeutic response in the animal over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend upon a varietyof factors including the strength of the particular compound employed,the condition of the animal, the body weight of the animal, as well asthe severity of the adverse condition or infection and stage of thecondition or disease. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound. Suitable dosesand dosage regimens can be determined by comparisons to agents presentlyused in the treatment of autoimmune disease and problems of graft-hostrejection. The preferred dosage is the amount which results in thedesired effect on T cell effector response, without significant sideeffects. In proper doses and with suitable administration of certaincompounds, the present invention provides for a wide range of alterationof T cell effector responses, e.g., from little alteration toessentially either complete induction or inhibition.

The following examples further illustrate the present invention but, ofcourse, should not be construed as in any way limiting its scope.

EXAMPLES

The following experimental procedures were employed in carrying out theexperiments which form the examples described herein.

cDNA Constructs and L Cell Transfectants

cDNA expression constructs encoding wild-type Eβ^(k) (kkk) and mutantchains including β75^(s),79^(s) (kss), β72^(s),75^(s),79^(s) (sss),β72^(s) (skk), β75^(s) (ksk) and β79^(s) (kks) were created as describedin Racioppi et al., J. Immunol., 147, 3718-27 (1991). These plasmidswere co-transfected into the DAP.3 subline of mouse L cells (Margulieset al., J. Immunol., 130, 463-70 (1983)) together with a constructencoding wild-type Eα and a plasmid containing a marker gene for drugselection. Drug-resistant clones expressing suitable surface levels ofEαEβ dimers were isolated as described in Racioppi et al., J. Immunol.,147, 3718-27 (1991).

Peptides

The CNBr fragment 81-104 from pigeon cytochrome c (PCC 81-104) wasprepared as described in Corradin et al., Biochim. Biophys. Acta., 221,489-496 (1970). DASP, an analogue of moth cytochrome c (residues 86-90linked to residues 94-103), as well as PCC 88-104, hen egg lysozyme(HEL) 81-96, and HEL 46-61with or without an NH₂ -terminal long-chainbiotin (Busch et al., Int. Immunol., 2, 443-51 (1990)) were synthesizedand purified by Dr. John Coligan, Biological Resources Branch, NIAID,NIH, Bethesda, Md. DASP conjugated to an NH₂ -terminal long-chain biotinwas prepared and kindly donated by Dr. Jonathan Rothbard, ImmuLogicCorp., Palo Alto, Calif. PCC 81-104 99Q! was the kind gift of Dr. RonaldSchwartz, LCMI, NIAID, NIH, Bethesda, Md.

T Cell Clones and Hybridomas

The 3C6 T cell clone was produced as described in Matis et al., J.Immunol., 130, 1527-35 (1983b) from pigeon cytochrome c-immune spleencells of a B10.A mouse. The C6E1 T cell hybridoma was produced from the3C6 clone by fusion to BW1100 (White et al., J. Immunol., 143, 1822-5(1989)), which lacks its own functionally rearranged TCR α and β geneloci. The hybridoma and the T cell clone were grown in RPMI-1640 mediumwith 10% fetal calf serum (FCS), 2 mM glutamine, and nonessential aminoacids. The 3C6 cytochrome c-specific T cell clone was maintained invitro by stimulation with antigen and irradiated spleen cells, followedby a period of rest in the absence of antigen. Cells collected at end ofthe resting phase were centrifuged over a Ficoll gradient and treatedwith a cocktail of I-A specific and I-E specific mAbs. Antibody-treatedT cells were negatively selected by sorting with magnetic beads (DYNAL,Norway) and an MPC-1 magnetic concentrator as recommended by thesupplier.

T Cell Functional Assays

Production of IL-2 and IL-3 in response to L cell transfectants in thepresence or absence of added peptide was measured as described inRonchese et al., Nature, 329, 254-6 (1987b). In brief, 2-5×10⁴ T cellswere incubated with 2-5×10⁴ transfected L cells in the wells of 96-wellflat bottom culture plates in 200 μl of complete medium with or withoutvarious concentrations of peptide antigen. Supernatants were collectedafter 24 hours and assayed for IL-2 content using CTLL indicator cells,or for IL-3 content using FDC.1 cells. IL-2 and IL-3 units werecalculated as the inverse of the dilution giving 50% of the maximum ³H-thymidine incorporation by CTLL (IL-2) or FDC.1 (IL-3) cells observedwith reference IL-2 and IL-3 preparations. Proliferative responses weremeasured by assessing ³ H-thymidine incorporation between 48-66 hrs ofculture. For experiments in which the data are expressed as "% max" or"% alloresponse", the actual absolute 100% responses were: IL-2alloresponse, 10-40 units/ml; IL-3 alloresponse, 100-500 units/ml; IL-2response to EαEβ^(k) plus PCC peptide, 150-300 units/ml; IL-3 responseto EαEβ^(k) plus PCC peptide, 1000-10,000 units/ml; IL-2 response tofixed cells bearing EαEβ^(k) plus PCC peptide, 30-50 units/ml; IL-3response to fixed cells bearing EαEβ^(k) plus PCC peptide, 50-200units/ml.

For analysis of the effects of mAb directed against CD28 on the responseof the 3C6 Th1 clone, 5×10⁴ 3C6 Th1 cells were co-cultured with 5×10⁴transfected L cells expressing the allostimulatory MHC molecule with ksschains in the presence or absence of varying dilutions (1:250-1:16,000)of ascites containing hamster anti-mouse CD28 mAb (Harding et al.,Nature, 356, 607-9 (1992)). IL-2 and IL-3 accumulation in these cultureswas measured as described above. Where indicated, CD28 was cross-linkedon the cell surface by first pre-incubating the 3C6 Th1 cells with themAb for 1 hour at 4° C., washing the cells, and then adding polyclonalanti-hamster IgG antisera (Caltag) to a final dilution of 1:40.Following incubation for 1 hour at 4° C., the cells were washed beforeuse in a standard stimulation culture.

Flow Cytometric Analysis

Analysis of cell-surface class II MHC molecule expression by transfectedcells was carried out using the anti-Eα mAb 14.4.4S (Ozato et al., J.Immunol., 124, 533-40 (1980)) and FITC-goat anti-mouse F(ab)'2 as thedetection reagent (Ronchese et al., J. Immunol., 139, 62-38 (1987a)).Cells were analyzed using either an EPICS V, FACSAnalyzer, or FACScan.For multiparameter analysis of 3C6 Th1 cells incubated with APC with orwithout peptide, 3C6 Th1 cells (0.5×10⁵) were co-cultured with FT27.2.A2 I-E expressing L cells (0.5×10⁵) that had been preincubated for1 hour at 37° C. with PCC 81-104 peptide (20 μM). After 24 hour ofincubation, cells recovered from the 48 well plate were stained using ananti-Thy 1.2 mAb labeled with FITC (Becton-Dickinson) and mAb 7D4, ananti IL2-Rα specific reagent (Malek et al., Proc. Natl. Acad. Sci. USA,80, 5694-8 (1983)). Detection of bound 7D4 was with R-PE labelled goatanti-rat antibody (Caltag). Cells were analyzed on a FAScan flowcytometer. Cells were gated on Thy 1.2 staining, and the positive cells(i.e., 3C6 Th1 cells) were analyzed for 7D4 expression and cell size(SSC side-scatter! parameter). Supernatants of these cultures wereassayed for IL-2 and IL-3 content. Parallel cultures were analyzed for ³H-thymidine incorporation.

Measurement of Peptide Binding Using Biotinylated Peptides

A modified version of the assay described in Busch et al., Int.Immunol., 2, 443-51 (1990), was used to measure peptide binding tocell-associated class II MHC molecules. After extensive washing inphosphate buffered saline (PBS)-1% bovine serum albumin (BSA), 2×10⁵DAP.3 cells or DAP.3 transfectants expressing various EαEβ moleculeswere incubated at 37° C. in 200 μl of PBS-2% FCS containing theindicated concentration of biotinylated peptide. In the competitionexperiments competitors were added at the same time as labeled peptideand at the indicated concentrations. After 4 hours, the cells werewashed and stained with a sandwich of FITC-avidin (Vector),biotinylated-anti-avidin (Vector), and FITC-avidin (Vector). Afterwashing, the cells were analyzed on a FAScan flow cytometer forfluorescence. Only viable cells were considered in the analysis, asdetermined by PI staining. The data are expressed as net meanfluorescence intensity (MFI), calculated by subtracting from the actualMFI the MFI obtained by staining in the absence of biotinylated-peptide.Percent inhibition in the competition experiments was calculated as: %of I=(net MFI without competitor-net MFI with competitor)/(net MFIwithout competitor×100%).

Measurement of Cytokine mRNA Levels

3C6 Th1 cells were co-cultured with FT 27.2.A2 cells expressing theallostimulatory mutant EαEβ class II molecule that had previously beenincubated with PCC 81-104 peptide for 1 hour at 37° C. After 4 hours ofco-culture, total RNA was extracted by the guanidinium thiocyanatephenol-chloroform method (Chomczynski et al., Anal. Biochem., 162, 156-9(1987)), quantified spectro-photometrically, and analyzed on adenaturing agarose gel. Reverse transcription of 1 μg of total RNA with37.5 μg/ml of an oligo d(T)₁₂₋₁₈ (Collaborative Research) was performedfor 1 hour at 42° C. using 600 U of M-MLV reverse transcriptase (GIBCOBRL) in 50 mM Tris-HCl, pH 8.3, 3 mM MgCl₂, 60 mM KCl, 10 mMdithiothreitol, 75 μg/ml of acetylated BSA, 1 unit/ml of RNasin(Promega) and 1 mM of each of dATP, dGTP, dCTP, dTTP. 1 μCi of ³² P-dCTP(3000 μCi/mM, Amersham) was added to the reaction mixture. Theefficiency of the reverse transcription reaction was assayed bycomparing the TCA-precipitable radiolabeled cDNA present in each sample.PCR analysis was performed on cDNA samples adjusted to contain the sameamounts of TCA-precipitable labeled material. One-tenth of the productcDNA was combined with 1 μM of each of the specific IL-2 and IL-3primers (Cytokine Mapping Amplimers®, Clontech), 200 μM of each dNTP,and 1.25 U of Taq DNA polymerase (5 U/ml, Perkin Cetus) in 1× PCR buffer(10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 100 μg/ml BSA). Totalvolume was 50 μl. PCR reactions were performed as described in theCytokine Mapping Amplimerst® manual (Clontech). The number of cycles wasadjusted to fall in the log linear range of signal for these amplimersand cDNA source. PCR products were analyzed on 5% polyacrylamidepre-cast TBE gels (Bio-Rad). Gels were dried and radiolabeled bands werequantified using a Phosphor-Imager (Molecular Dynamics). Images wereanalyzed by Image Quant® version 3.15 software (Molecular Dynamics).

Example 1

This example sets forth a mutant peptide-MHC class II molecule complexthat is a TCR ligand in accordance with the present invention andillustrates peptide inhibition of alloreactive stimulation of IL-2production in a T cell clone by the TCR ligand.

3C6 is a Th1-type (Mosmann et al., J. Immunol., 136, 2348-57 (1986))cloned cell line derived from a pigeon cytochrome c (PCC)-immunizedB10.A mouse that increases its production of IL-2 when stimulated bysplenic APC as well as C-terminal PCC peptides including the CNBrfragment 81-104 (PCC 81-104). In addition to the expected specificactivation of 3C6 Th1 cells by PCC peptides presented by cellsexpressing the wild-type EαEβ^(k) MHC class II molecule, 3C6 Th1 cellsalso respond alloantigenically to APC expressing the closely relatedEαEβ^(s) molecule. This alloantigenic response can be obtained in theabsence of added antigen, which has been observed for other cytochromec-reactive T cells (Matis et al., J. Immunol., 130, 1527-35 (1983b)), aswell as for other alloresponses.

Transfected mouse L cells expressing MHC class II molecules with mutantEβ^(k) proteins containing various Eβ^(s) allelic substitutions wereproduced to examine the relationships among MHC molecule structure,peptide antigen presentation, and allorecognition (Racioppi et al., J.Immunol., 147, 3718-27 (1991)). Transfectants were examined thatexpressed EαEβ MHC molecules with wild-type Eβ chains (kkk), and Eβchains containing substitutions of E^(s) allelic residues at positions75 and 79 (kss), at positions 72, 75, and 79 (sss), at position 72(skk), at position 75 (ksk), and at position 79 (kks) in the putativehelix of the peptide-binding region.

While tranfectants expressing MHC molecules with wild-type (kkk) chains,or chains possessing a single allelic substitution (skk, ksk, kks)failed to evoke stimulation of IL-2 synthesis in 3C6 Th1 cells in theabsence of peptide, transfectants expressing MHC molecules with chainspossessing multiple allelic substitutions (sss, kss) evoked asubstantial peptide-independent, alloantigenic response (FIG. 1A).

3C6 Th1 was then tested for its response to PCC 81-104 peptide presentedon the APC in association with the two types of MHC molecules withmutant Eβ chains (i.e., sss and kss) capable of mediating alloreactivestimulation of IL-2 production. In addition to inducing apeptide-independent alloresponse, a clear dose-dependent stimulation ofIL-2 production by PCC 81-104 peptide was observed with APC expressingMHC molecules with sss chains (FIG. 1B). Unexpectedly, addition of thesame peptide to cells expressing MHC molecules with kss chains not onlyfailed to stimulate IL-2 production above that seen in the absence ofpeptide, but decreased IL-2 levels with increasing peptide doses tolevels below those observed in the alloantigenic response (FIG. 1B).

Example 2

This example confirms that the PCC peptide-mediated inhibition ofalloreactive stimulation of IL-2 production in 3C6 Th1 cellsdemonstrated in Example 1 cannot be explained by competition with otherpeptides for binding with MHC molecules possessing kss chains.

Substantial evidence supports that many T cell responses toalloantigenic MHC molecules involve recognition of the peptide(s) boundto the non-self MHC molecules (Heath et al., Nature, 341, 749-52 (1989);Lombardi et al., J. Immunol., 142, 753-9 (1989); Cotner et al., J.Immunol., 146, 414-7 (1991); Rotzschke et al., J. Exp. Med., 174,1059-71 (1991)). Thus, one explanation for the ability of the PCC 81-104peptide to inhibit the 3C6 Th1 alloresponse to the mutant MHC class IImolecule with kss chains would be that it competes for MHC binding withanother peptide that is necessary for the formation of the alloantigenicligand recognized by the 3C6 Th1 receptor. Competition at the level ofthe MHC molecule has been suggested as an explanation for previousobservations of exogenous peptide inhibition of allogeneic stimulation(Rock et al., J. Exp. Med., 159, 1238-52 (1984); Eckels et al., Proc.Natl. Acad. Sci. USA, 85, 8191-5 (1988); Wei et al., J. Exp. Med., 174,945-8 (1991)). However, it is usually difficult to block MHC-dependentresponses by adding a competing peptide after the stimulatory peptidehas had an opportunity to bind (Maryanski et al., J. Exp. Med., 167,1391-405 (1988); Adorini et al., Nature, 342, 800-3 (1989)). In thepresent case, if the culture medium or the transfectant itself were thesource of the putative peptide needed for allostimulation, then culturedcells would have had ample time to form the stimulatory peptide-MHCmolecule complexes prior to the introduction of the potentiallycompeting PCC 81-104 peptide.

Possible competition for peptide-MHC binding by PCC 81-104 wasinvestigated by examining and comparing the effects of peptides PCC81-104, PCC 99Q!, HEL 46-61, and DASP on 3C6 Th1 alloreactivestimulation of IL-2 production. All peptides with the exception of DASPwere able to bind the MHC molecule with kss chains to a similar extentas the inhibitory PCC 81-104 peptide (FIG. 2A). Despite this similarityin MHC molecule binding, the peptides varied over two to three orders ofmagnitude in their ability to stimulate production of IL-2 in 3C6 Th1cells when presented by the wild-type EαEβ^(k) molecule (FIG. 2B).Whereas comparable results were obtained with PCC 81-104 and DASP, PCC99Q! demonstrated impaired ability to stimulate IL-2 production. Thesedata confirm the assignment of position 99 as a key epitopic residue inthe PCC determinant (Hansburg et al., J. Immunol., 131, 319-24 (1983);Fox et al., J. Immunol., 139, 1578-88 (1987); Jorgensen et al., Nature,355, 224-30 (1992)), and show that the change at this position fromlysine to glutamine affects TCR-dependent recognition, and not MHCmolecule binding. Moreover, the ability of the peptides to inhibit the3C6 Th1 alloresponse to MHC molecules with kss chains was directlyrelated to their capacity to stimulate the clone in the context of thewild-type EαEβ^(k) molecule (FIG. 2C). Namely, PCC 81-104 peptide, whichshowed the greatest ability to stimulate IL-2 production when presentedby wild-type MHC molecules also showed the greatest ability to inhibitthe 3C6 Th1 alloresponse to the MHC molecules with kss chains.Similarly, PCC 99Q! peptide, which showed the least ability to stimulateIL-2 production when presented by wild-type MHC molecules also showedthe least ability to inhibit the 3C6 Th1 alloresponse to MHC moleculeswith kss chains. Thus, it is the fine specificity of the 3C6 Th1 TCR forthe peptide, and not the ability of the peptide to bind to the mutantMHC molecule which dictates capacity to inhibit alloantigen-stimulatedIL-2 production. This argues against competition between peptides forMHC binding as the cause of peptide inhibition of alloreactivity.

Additional experiments using an unrelated hen egg lysozyme (HEL 81-96)instead of PCC peptide support this conclusion (FIG. 3). Although HEL81-96 bound well to the MHC molecules with kss chains (FIG. 3A), thispeptide, like PCC 99Q!, lacked ability to inhibit alloreactivestimulation of IL-2 production in 3C6 Th1 cells (FIG. 3B).

Example 3

This example confirms that the PCC peptide-mediated inhibition ofalloreactive stimulation of IL-2 production in 3C6 Th1 cellsdemonstrated in Example 1 cannot be explained by high-dose suppression.

Mouse Th1 clones including 3C6 Th1 show a characteristic decline inantigen-stimulated proliferation as the concentration of offered antigenis increased to high levels (Matis et al., Proc. Natl. Acad. Sci. USA,80, 6019-23 (1983a); Suzuki et al., J. Immunol., 140, 1359-65 (1988)).This high-dose suppression appears to be related to prolonged orrepetitive engagement of the TCR. The possibility that the low apparentresponse of 3C6 Th1 cells to MHC molecules with kss chains was due to ahigh level of allostimulation, and was attenuated by additional PCCpeptide-MHC molecule complexes was investigated.

Upon stimulation of 3C6 Th1 cells with wild-type MHC molecules plus PCC81-104 peptide, high-dose suppression of proliferation was observed,whereas stimulation of IL-2 production increased with increasing amountsof added peptide (FIG. 4A). The 3C6 Th1 cells showed a different.phenotype upon stimulation with MHC molecules possessing kss chains plusPCC 81-104 peptide (FIG. 4B). In this case, increasing amounts of addedPCC peptide decreased IL-2 accumulation in a dose-dependent fashion andonly marginally diminished proliferation. Furthermore, addition ofanti-EαEβ mAb to 3C6 Th1 stimulated with MHC molecules possessing ksschains did not result in augmentation of IL-2 production in the absenceof PCC peptide (data not shown), which would be expected due toreduction in receptor engagement caused by antibody addition if thealloresponse were in the high-dose suppression range. Thus, theseresults verify that the inhibitory effect of peptide PCC 81-104 is notdue to excessive TCR-dependent stimulation of 3C6 Th1.

Example 4

This example confirms that the PCC peptide-mediated inhibition ofalloreactive stimulation of IL-2 production in 3C6 Th1 cellsdemonstrated in Example 1 is specific for this effector response anddoes not inhibit all TCR-dependent signalling.

Several experiments were performed to determine if TCR-dependent signaltransduction occurs when 3C6 Th1 cells are exposed to MHC molecules withkss chains plus PCC 81-104 peptide. Even though PCC 81-104 peptideaddition inhibited alloreactive stimulation of IL-2 accumulation (IL-2)in 3C6 Th1 cells, TCR-dependent alloreactive IL-2Rα production (IL-2Rα),cell size enlargement (SSC) and IL-3 production (IL-3) proceeded as theydid in the absence of added PCC peptide (data shown pictorially in FIG.5A and quantitatively in FIG. 5B). Thus, 3C6 Th1 continued to receivebiologically active signals through the TCR under conditions in whichthe IL-2 alloresponse was abrogated. This validates that the activity ofcomplexes of PCC 81-104 with mutant MHC molecules possessing kss chainsin inhibiting production of IL-2 did not result from the elimination ofeffective TCR occupancy by the agonist alloantigen (FIGS. 5C and 5D).

The decrease in IL-2 in 3C6 Th1 cells upon presentation of peptide byMHC molecules with kss chains was not the result of increased IL-2consumption by proliferating cells, but was due to a decrease insteady-state IL-2 mRNA levels. This decrease was specific for IL-2 mRNA,as IL-3 mRNA levels were similar in the presence or absence of added PCCpeptide.

Example 5

This example supports that the PCC peptide-mediated inhibition ofalloreactive stimulation of IL-2 production in 3C6 Th1 cellsdemonstrated in Example 1 may arise from interference with selectivesynergy between co-stimulatory and TCR-generated signals, and not from areduction in the number of TCR available for alloantigen recognition dueto engagement of TCR by PCC peptide-mutant MHC molecule complexes andfailure to contribute to TCR-dependent signal generation.

The inhibitory effect of PCC peptide on the 3C6 Th1 alloresponse is notdue to inhibition of alloantigen formation or high-dose suppression(Examples 2 and 3). While inhibition is related to receptor engagementby the PCC peptide-mutant MHC molecule complexes (Example 2), it doesnot result from a blockade of all TCR-dependent signal transduction(Example 4). The further less-likely possibility that the PCCpeptide-mutant EαEβ complexes reduced the number of TCR available foralloantigen recognition by engaging the TCR without contributing tosignal generation was investigated by examining the relative sensitivityof 3C6 Th1 cells to stimulation of IL-2 and IL-3 production withwild-type MHC molecules plus PCC 81-104 peptide. As can be seen in FIG.6, 100-fold less peptide was required to attain the same fraction ofmaximal response for IL-2 production than was needed for IL-3production. This suggests that IL-3 production requires higher, notlower, TCR occupancy than does IL2 production. This is the opposite ofwhat would be required to explain selective peptide-MHC molecule complexdependent inhibition of alloantigen-driven IL-2 but not IL-3 productionon the basis of differential sensitivity to residual TCR signalling,because at any level of reduced occupancy based on TCR blockade, agreater reduction in IL-3 than IL-2 production should have been seen.

The phenotype of TCR-dependent IL-3 production in the absence of IL-2production by Th1 clones has been previously reported. Namely, Th1clones stimulated by peptide-MHC molecule complexes in planar membranesgave significant, though subnormal IL-3 production, whereas IL-2production was not observed under these conditions (Quill et al., J.Immunol., 138, 3704-12 (1987)). The defect in IL-2 production underthese conditions has been attributed to the absence of at least oneessential co-stimulatory signal provided by viable APC which is separateand distinguishable from ligand-receptor interactions that affect theoccupancy of the TCR or its production of second messengers (Mueller etal., Ann. Rev. Immunol., 7, 445-80 (1989b)). The possibility in thepresent case that the ability of 3C6 Th1 cells to produce IL-2 at lowligand densities with viable APC resulted from a requisite synergybetween TCR signalling and co-stimulatory signalling that was not neededfor IL-3 gene activation was investigated by comparing thedose-responses for the two cytokines using aldehyde-fixed APC withgreatly reduced levels of co-stimulatory signals. As previously reported(Jenkins et al., J. Exp. Med., 165, 302-19 (1987)), the cytokineresponses elicited by fixed APC were reduced more than 90% as comparedwith live APC (data not shown). However, the antigen concentrationneeded for half-maximal IL-3 responses with fixed APC was only slightlyincreased (about three-fold) compared to that needed with viable APC,whereas the dose needed for half-maximal IL-2 production with fixed APCincreased close to 100-fold to approach that required for IL-3 (FIG. 6).

These results confirm that peptide inhibition of alloreactivestimulation of IL-2 production in 3C6 Th1 cells is the result of anecessary synergy between TCR-generated signals and co-stimulatorysignals.

Example 6

This example further validates a role for co-stimulatory signalling inthe PCC peptide-mediated inhibition of alloreactive stimulation of IL-2production in 3C6 Th1 cells demonstrated in Example 1.

Cytokine responses to viable and fixed cells described in Example 5confirm that the selective inhibition by PCC-mutant MHC moleculecomplexes of alloantigen-stimulated IL-2 production may reflectinterference with the generation of, or response to, co-stimulation in3C6 Th1 cells. The L cell transfectant shown in FIG. 6 which expresseswild-type MHC molecules also constitutively expresses the membraneprotein B7 (Razi-Wolf et al., Proc. Natl. Acad. Sci. USA, 89, 4210-14(1992)), which is a ligand for the CD28 co-stimulatory pathway. Thecapacity to observe low but detectable IL-2 production using this L celltransfectant as an APC after it has been fixed seems to relate to a lowlevel of residual co-stimulation that is mediated by pre-existing B7protein present on the fixed cells. Since experiments with purifiedpeptide-MHC molecule complexes in planar membranes demonstrate that Th1clones do not produce IL-2 in response to peptide-MHC molecule ligandsin the absence of co-stimulatory signals (Quill et al., J. Immunol.,138, 3704-12 (1987)), alloantigen-mediated 3C6 Th1 IL-2 production inthe complete absence of co-stimulation could not be investigated.However, since T cell hybridomas do produce IL-2 in the absence ofco-stimulatory signals (Watts et al., Proc. Natl. Acad. Sci. USA, 81,7564-8 (1984)), the effect of PCC peptide on the alloreactivestimulation of IL-2 production of a T cell hybridoma bearing the sameTCR as 3C6 Th1 was investigated. This T cell hybridoma was derived byfusion of a TCR-chain negative T lymphoma cell with the 3C6 Th1 clone,and is characterized by a peptide and alloantigenic response profilethat is indistinguishable from the 3C6 Th1 clone (data not shown).

Unlike the 3C6 Th1 clone, in the presence of viable APC, the hybridomadid not exhibit a substantial difference in its IL-2 and IL-3dose-responses to PCC 81-104 antigen presented by wild-type MHCmolecules with kkk chains (FIG. 7A). Instead, the hybridoma responded toviable APC almost precisely as did the 3C6 Th1 clone to fixed APClacking almost all co-stimulatory activity. This suggests thatco-stimulation related signals do not play a major role in IL-2production by the hybridoma. The results also support that thedifference in the cytokine dose-responses to antigen presented by fixedor viable cells seen for 3C6 Th1 was the result of the essentialcontribution of co-stimulation to the IL-2 response of normal T cells.The alloantigen-stimulated IL-2 response of the hybridoma was notdecreased by addition of PCC peptide (FIG. 7B), which further supportsthat the inhibitory effect of PCC peptide on the alloresponse of the 3C6Th1 cells was due to interference with exogenously providedco-stimulation.

These data also confirm that the complexes of PCC peptide with the MHCmolecules possessing kss chains are not complete antagonists of, but areactually weak agonists for, TCR signal generation, since at high antigenconcentrations they could clearly elicit an effector response from thehybridoma, as evidenced by increased IL-3 production. Given the slightdose-response advantage seen for IL-2 production over IL-3 productionupon peptide presentation by wild-type MHC molecules (FIG. 7A), it issomewhat surprising that a stimulation of IL-2 production was notobserved with antigen presented by MHC molecules with kss chains.However, this may be due to generation of qualitatively differentsignals upon TCR engagement of wild-type MHC-PCC peptide complexes ascompared with mutant MHC-PCC peptide ligand, and the mutant MHC-PCCpeptide ligand may simply be inadequate to evoke IL-2 responses in thehybridoma. Alternatively, even though co-stimulation is not required forIL-2 production by hybridomas, it modestly increases such responses andthus could explain the slight dose-response advantage seen for IL-2compared to IL-3.

Example 7

This example supports that the PCC peptide-mediated inhibition ofalloreactive stimulation of IL-2 production in 3C6 Th1 cellsdemonstrated in Example 1 either involves interference withco-stimulatory signal transduction within the T cell or the disruptionof a crucial distinct co-stimulatory pathway.

Interaction of the CD28 molecule on T cells with the B7 membrane proteinon APC appears to activate a major co-stimulatory pathway involved inregulating IL-2 production (Linsley et al., J. Exp. Med., 173, 721-30(1991); Koulova et al., J. Exp. Med., 173, 759-62 (1991); Freeman etal., J. Exp. Med., 174, 625-31 (1991); Gimmi et al., Proc. Natl. Acad.Sci. USA, 88, 6575-9 (1991); Reiser et al., Proc. Natl. Acad. Sci. USA,89, 271-5 (1992); Vandenberghe et al., J. Exp. Med., 175, 951-60(1992)). Antibody to the CD28 molecule on mouse or human T cells canmodulate the cytokine response of such cells to TCR stimulation (Damleet al., J. Immunol., 140, 1753-1761 (1988); Ledbetter et al., Blood, 75,1531-9 (1990); Harding et al., Nature, 356, 607-9 (1992)), presumably byaltering delivery of a critical co-stimulatory signal. Because theobservations described herein suggested that the PCC-induceddownregulation of IL-2 production was related to the necessity ofco-stimulation for IL-2 allostimulation, the role of CD28 in IL-2responses of the 3C6 Th1 clone and the possibility thatantibody-mediated activation of the CD28 signalling pathway mightcounteract the inhibitory effect of the peptide-mutant MHC moleculecomplexes was examined.

Inclusion of soluble anti-CD28 in a co-culture of 3C6 Th1 cells and theL cell transfectant expressing MHC molecules with kss chains resulted inalmost complete inhibition of IL-2 production, and did not decrease IL-3production (FIG. 8A). This is consistent with prior data on the effectof soluble anti-CD28 on IL-2 secretion in response to alloantigen (Damleet al., J. Immunol., 140, 1753-1761 (1988)) and replicates the phenotypeseen upon addition of PCC peptide to similar cultures. The ability ofsoluble anti-CD28 to inhibit alloreactive stimulated IL-2 production bythe 3C6 Th1 clone supports a critical role for CD28 in co-stimulation ofIL-2 production, presumably via interaction with the B7 surface proteinpresent on the transfected L cells.

Antibody-mediated cross-linking of CD28 resulted in a small increase inIL-3 production consistent with the previously described ability ofco-stimulation to augment this response without being required for it.Antibody-mediated cross-linking of CD28 nullified the inhibitory effectof soluble anti-CD28 on alloreactive stimulation of IL-2 production in3C6 Th1 cells. Thus, even though results obtained with soluble mAbverified the necessity of CD28-dependent signalling for IL-2 productionby 3C6 Th1 cells, stimulation of this pathway by cross-linking CD28 didnot reverse the inhibition of IL-2 secretion mediated by PCC peptideaddition (FIG. 8B). These results confirm that peptide-mediatedinhibition of IL-2 secretion involves either interference with effectiveCD28 co-stimulatory signal transduction within the T cell or disruptionof a crucial co-stimulatory pathway distinct from that evoked by CD28aggregation, such as a pathway involving heat-stable antigen (Kay etal., J. Immunol., 145, 1952-59 (1990)), which has recently beensuggested as another APC-expressed molecule regulating IL-2 production(Liu et al., J. Exp. Med., 175, 437-45 (1992)).

All of the references cited herein are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon apreferred embodiment, it will be obvious to those of ordinary skill inthe art that variations of the preferred compounds and methods may beused and that it is intended that the invention may be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications encompassed within the spirit andscope of the invention as defined by the following claims.

What is claimed is:
 1. A method of identifying an altered T cellreceptor ligand that substantially inhibits at least one T cell effectorresponse, said altered T cell receptor ligand comprising an MHC moleculeand a peptide, wherein the MHC molecule is altered by mutation or thepeptide is altered by the replacement of at least one residue, whichmethod comprises contacting a T cell with an agonist T cell receptorligand capable of effecting a known T cell effector response and acandidate altered T cell receptor ligand and determining whether saidcandidate altered T cell receptor ligand substantially inhibits at leastone T cell effector response while not substantially inhibiting at leastone other T cell effector response.
 2. The method of claim 1 whereinsaid T cells are simultaneously contacted with said agonist T cellreceptor ligand and said candidate altered T cell receptor ligand bycontacting said T cells with a mixture of said agonist T cell receptorligand and said candidate altered T cell receptor ligand.
 3. The methodof claim 2, wherein said mixture of said agonist and said candidatealtered T cell receptor ligand is formed by contacting MHC moleculeswith a first peptide to form said agonist and then with a second peptideto form said candidate altered T cell receptor ligand.
 4. The method ofclaim 1, wherein said T cells are contacted with said agonist and thensaid T cells and said agonist are contacted with said candidate alteredT cell receptor ligand.
 5. A method of preparing and identifying analtered T cell receptor ligand that substantially inhibits at least oneT cell effector response, wherein said altered T cell receptor ligandcomprises an MHC molecule and a peptide, and wherein peptide is alteredby the replacement of at least one residue, which method comprises:(a)identifying an agonist T cell receptor ligand comprising an unalteredpeptide which binds to an unaltered MHC molecule to form a complex thatcan evoke T cell effector responses; (b) preparing altered peptides bydetermining by substitution those amino acids which, when present ateach position of said peptide do not prevent formation of thepeptide:MHC complex; (c) binding each of the altered peptides tounaltered MHC molecules to form candidate altered T cell receptorligands; (d) screening said candidate altered T cell receptor ligands toidentify peptide-MHC complexes that have reduced or distinct agonisticeffects as compared to the agonist effects of the peptide-MHC moleculecomplex comprising the unaltered T cell receptor ligand; and (e)screening each candidate altered T cell receptor ligand identified instep (d) for the ability to inhibit at least one T cell effectorresponse typically elicited by unaltered agonist T cell receptor ligandalone, by contacting the T cell with both said candidate altered T cellreceptor ligand and unaltered T cell receptor ligand.
 6. The method ofclaim 5, which method further comprises determining whether saidcandidate T cell receptor ligand substantially inhibits at least one Tcell effector response while not substantially inhibiting at least oneother T cell effector response.
 7. The method of any one of claims 1-6,wherein the MHC molecule of said altered T cell receptor ligand is aClass I MHC molecule.
 8. A method of preparing and identifying analtered T cell receptor ligand that substantially inhibits at least oneT cell effector response, but not all T cell effector responses, whereinsaid altered T cell receptor ligand comprises a MHC molecule and apeptide, and wherein the MHC molecule is altered by mutation, whichmethod comprises:(a) identifying an agonist T cell receptor ligandcomprising an unaltered peptide and an unaltered MHC molecule that formsa complex and can evoke T cell effector responses; (b) obtaining mutantforms of said MHC molecule, wherein said mutants do not preventformation of the peptide:MHC complex; (c) binding the unaltered peptideto said mutant forms of said MHC molecule to form candidate altered Tcell receptor ligands; (d) screening said candidate altered T cellreceptor ligands to identify ligands that have reduced or distinctagonistic effects as compared to the agonist effects of the agonist Tcell receptor ligand; and (e) screening each candidate altered T cellreceptor ligand identified in step (d) for the ability to inhibit atleast one T cell effector response, but not all T cell effectorresponses, typically elicited by the agonist T cell receptor ligandalone, by contacting a T cell with both said candidate altered T cellreceptor ligand and said agonist T cell receptor ligand.
 9. A method ofpreparing and identifying an altered T cell receptor ligand thatstimulates at least one T cell effector response, but not all T celleffector responses, wherein said altered T cell receptor ligandcomprises a MHC molecule and a peptide, and wherein the MHC molecule isaltered by mutation, which method comprises:(a) identifying an agonist Tcell receptor ligand comprising an unaltered peptide and an unalteredMHC molecule that forms a complex and can evoke T cell effectorresponses; (b) obtaining mutant forms of said MHC molecule, wherein saidmutant forms do not prevent formation of the peptide:MHC complex; (c)binding the unaltered peptide to said mutant forms of said MHC moleculeto form a candidate altered T cell receptor ligand; (d) screening saidcandidate altered T cell receptor ligand to identify ligands that havereduced or distinct agonistic effects as compared to the agonist effectsof the agonist T cell receptor ligand; and (e) screening each candidatealtered T cell receptor ligand identified in step (d) for the ability tostimulate at least one T cell effector response, but not all T celleffector responses, typically elicited by the agonist T cell receptorligand alone, by contacting a T cell with said candidate altered T cellreceptor ligand.